The present invention pertains to compositions related to proteins which function in controlling biology and physiology of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides purified genes, proteins, antibodies, related reagents, and methods useful, e.g., to regulate activation, development, differentiation, and function of various cell types, including hematopoietic cells.
Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the xe2x80x9cimmune networkxe2x80x9d. See, e.g., Paul (1998) Fundamental Immunology (4th ed.) Raven Press, NY. Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which will lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system disorders. Some of these factors are hematopoietic growth factors, e.g., granulocyte colony stimulating factor (G-CSF). See, e.g., Thomson (ed. 1998) The Cytokine Handbook (3d ed.) Academic Press, San Diego; Mire-Sluis and Thorpe (ed. 1998) Cytokines Academic Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating Factors Cambridge University Press; and Aggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and/or differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T-cells) making up the immune network. These lymphocytes interact with many other cell types.
Research to better understand and treat various immune disorders has been hampered by the general inability to maintain cells of the immune system in vitro. Immunologists have discovered that culturing these cells can be accomplished through the use of T-cell and other cell supernatants, which contain various growth factors, including many of the lymphokines.
From the foregoing, it is evident that the discovery and development of new lymphokines, e.g., related to G-CSF and/or IL-6, could contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve the immune system and/or hematopoietic cells. In particular, the discovery and development of lymphokines which enhance or potentiate the beneficial activities of known lymphokines would be highly advantageous. The present invention provides new interleukin compositions and related compounds, and methods for their use.
The present invention is directed to mammalian, e.g., primate or rodent, interleukin-B60 (IL-B60) (SEQ ID NO:1 and 2 (human); SEQ ID NO:3 and 4 (murine)) and its biological activities. It includes nucleic acids coding for polypeptides themselves and methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to complementary DNA (cDNA) sequences disclosed herein, and/or by functional assays for growth factor- or cytokine-like activities, e.g., G-CSF (see Nagata (1994) in Thomson The Cytokine Handbook 2d ed., Academic Press, San Diego) and/or IL-6 (see Hirano (1994) in Thomson The Cytokine Handbook 2d ed., Academic Press, San Diego). Also provided are polypeptides, antibodies, and methods of using them, including using nucleic acid expression methods. Methods for modulating or intervening in the control of a growth factor dependent physiology or an immune response are provided.
The present invention is based, in part, upon the discovery of a new cytokine sequence exhibiting significant sequence and structural similarity to G-CSF and IL-6. In particular, it provides primate, e.g., human, and rodent, e.g., mouse, genes encoding a protein whose mature size is about 198 amino acids. Functional equivalents exhibiting significant sequence homology will be available from other mammalian, e.g., cow, horse, and rat species.
Moreover, the present invention identifies a second associated component of a complex. Compositions related to the combination of components in the complex are provided, along with methods of use.
In one embodiment, the invention provides a substantially pure or recombinant polypeptide comprising the mature protein portion of SEQ ID NO: 2 or 4. Preferably, the polypeptide is: detectably labeled; unglycosylated; denatured; attached to a solid substrate; conjugated to another chemical moiety; or in a sterile composition. Kit forms include those comprising the polypeptide and: a compartment comprising the polypeptide; or with instructions for use or disposal of reagents in the kit.
Binding compounds include those-comprising an antigen binding site from an antibody that specifically binds to the described polypeptide. The binding compound can also be in a kit comprising: a compartment comprising the binding compound; or with instructions for use or disposal of reagents in the kit.
The invention further provides a method of producing an antigen:antibody complex, comprising contacting, under appropriate conditions, a primate IL-B60 polypeptide (SEQ ID NO:2) with an antibody that specifically or selectively binds the polypeptide of the invention, thereby allowing the complex to form.
Nucleic acid embodiments include an isolated or recombinant polynucleotide encoding the mature protein portion of SEQ ID NO: 2 or 4.
In other embodiments, the invention provides an isolated soluble complex comprising the mature protein portion of SEQ ID NO: 2 or 4, and the mature protein portion of SEQ ID NO: 12 or 13. Preferably the complex: comprises a recombinant polypeptide of SEQ ID NO: 2, 4, 12, or 13; is detectably labeled; is in a buffered solution; is in a sterile solution. Kits are provided containing such a complex and: a compartment comprising the complex; or instructions for use or disposal of reagents in the kit.
Binding compounds are provided comprising an antigen binding site from an antibody that specifically binds to the soluble complex but not to the mature polypeptide of SEQ ID NO: 12 or 13. Kits are provided comprising the binding compound and: a compartment comprising the binding compound; or instructions for use or disposal of reagents in the kit.
Methods are provided, e.g., of producing an antigen:antibody complex, comprising contacting, under appropriate conditions, a primate complex comprising IL-B60 (SEQ ID NO:2) and CLF-1 polypeptides (SEQ ID NO:12) with an antibody that selectively or specifically binds to an isolated soluble complex comprising the mature protein portion of SEQ ID NO: 2 or 4, and the mature protein portion of SEQ ID NO; 12 or 13, thereby allowing the complex to form.
Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding the mature protein portion of SEQ ID NO: 2 or 4, and the mature protein portion of SEQ ID NO: 12 or 13.
The invention also provides a composition of matter selected from: an isolated polypeptide comprising at least seven amino acids identical to segments of SEQ ID NO: 2 or 4; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 2 or 4; a natural sequence polypeptide comprising mature SEQ ID NO: 2 or 4; or a fusion polypeptide comprising IL-B60 (SEQ ID NO:2 or 4) sequence. In certain embodiments, the distinct nonoverlapping segments of identity include: one of at least eight amino acids; one of at least five amino acids and a second of at least six amino acids; at least three segments of at least four, five, and six amino acids, or one of at least twelve amino acids. In other embodiments the polypeptide of the composition of matter: is the polypeptide which: comprises a mature sequence of Table 1; is an unglycosylated form of IL-B60 (SEQ ID NO:2 or 4); is from a primate, such as a human; comprises at least seventeen amino acids of SEQ ID NO: 2 or 4; exhibits at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 2 or4; is a natural allelic variant of IL-B60 (SEQ ID NO: 2, 4); has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate IL-B60 (SEQ ID NO: 2); is glycosylated; has a molecular weight of at least 30 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; is a deletion or insertion variant from a natural sequence; or which further comprises: at least seven amino acids identical to segments of SEQ ID NO: 12 or 13; at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 12 or 13; a natural sequence polypeptide comprising mature SEQ ID NO: 12 or 13; or a primate CLF-1 (SEQ ID NO: 12). In additional preferred embodiments, the composition comprises: a substantially pure IL-B60 (SEQ ID NO: 1, 2, 3, or 4) and CLF-1 (SEQ ID NO: 12 or13); a sterile IL-B60 polypeptide (SEQ ID NO: 2 or 4) comprising the mature protein of SEQ ID NO: 2 or 4; or the described polypeptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration. The invention provides fusion polypeptides which comprise: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another cytokine receptor family protein, including CLF-1 (SEQ ID NO: 12). Kit embodiments include those comprising the polypeptide of the composition and: a compartment comprising the protein or polypeptide; or instructions for use or disposal of reagents in the kit.
The invention further provides methods of using the described polypeptides: to label the polypeptide, comprising labeling the polypeptide with a radioactive label; to separate the polypeptide from another polypeptide in a mixture, comprising running the mixture on a chromatography matrix, thereby separating the polypeptides; to identify a compound that binds selectively to the polypeptide, comprising incubating the compound with the polypeptide under appropriate conditions; thereby causing the component to bind to the polypeptide; or to conjugate the polypeptide to a matrix, comprising derivatizing the polypeptide with a reactive reagent, and conjugating the polypeptide to the matrix.
Related binding compounds include those comprising an antigen binding site from an antibody that specifically or selectively binds to a natural polypeptide, as described above, wherein: the binding compound is in a container; the IL-B60 polypeptide is from a human; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody; is raised against a mature polypeptide of Table 1; is raised against a mature IL-B60 (SEQ ID NO:2 or 4); is raised to a purified human IL-B60 (SEQ ID NO: 2); is immunoselected; is a polyclonal antibody; binds to a denatured IL-B60 (SEQ ID NO: 2 or 4); exhibits a Kd to antigen of at least 30 xcexcM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. Kits are provided comprising such a binding compound and: a compartment comprising the binding compound; or instructions for use or disposal of the reagents of the kit.
Methods are provided for producing an antigen:antibody complex, comprising contacting, under appropriate conditions, a primate IL-B60 polypeptide (SEQ ID NO: 2) with a described antibody, thereby allowing the complex to form. Preferably, in the method: the complex is purified from other cytokines; the complex is purified from other antibody; the contacting is with a sample comprising a cytokine; the contacting allows quantitative detection of the antigen; the contacting is with a sample comprising the antibody; or the contacting allows quantitative detection of the antibody.
In another embodiment the invention includes a composition comprising: a sterile binding compound, as described, or the binding compound and a carrier, wherein the carrier: wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding the described polypeptide, wherein: the IL-B60 is from a human (SEQ ID NO: 1 or 2); or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; encodes a plurality of antigenic peptide sequences of Table 4; exhibits Identity over at least thirteen nucleotides to a natural cDNA encoding the segment: is an expression vector; further comprises an origin of replication; is from a natural source: comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding the IL-B60 (SEQ ID NO: 2 or 4); or is a PCR primer, PCR product, or mutagenesis primer. Preferred embodiments include where the isolated or recombinant nucleic acid is in a cell or tissue. The cell maybe: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
Kits are provided comprising the described nucleic acid and: a compartment comprising the nucleic acid; a compartment further comprising a primate IL-B60 polypeptide (SEQ ID NO: 2); or instructions for use or disposal of reagents in the kit.
The invention further provides methods for forming a duplex with a polynucleotide described above, comprising contacting the polynucleotide with a probe that hybridizes, under stringent conditions, to at least 25 contiguous nucleotides of the coding portion of SEQ ID NO: 1, 3, or encoding the nature SEQ ID NO: 12 or 13; thereby forming the duplex.
In a further aspect, the invention provides a nucleic acid which: hybridizes under wash conditions of 30 minutes at 30xc2x0 C. and less than 2M salt to the coding portion of SEQ ID NO: 1; or exhibits Identity over a stretch of at least about 30 nucleotides to a primate IL-B60 (SEQ ID NO: 1). In preferred embodiments, the wash conditions that are at 45xc2x0 C. and/or 500 mM salt; or at 55xc2x0 C. and/or 150 mM salt; or the stretch is at least 55 nucleotides, e.g., at least 75 nucleotides.
Methods are provided, e.g., of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a mammalian IL-B60 (SEQ ID NO: 1, 2, 3, or 4); or contacting the cell with an agonist or antagonist of a complex comprising mammalian IL-B60 (SEQ ID NO: 2 or 4) and CLF-1 (SEQ ID NO: 12 or 13). Additionally, the invention provides a method of increasing the secretion of: an IL-B60 (SEQ ID NO: 2 or 4) sequence, comprising expressing the polypeptide with CLF-1 (SEQ ID NO: 12 or 13); or a CLF-1 (SEQ ID NO: 12 or 13), comprising expressing the CLF-1 (SEQ ID NO: 12 or 13) with an IL-B60 (SEQ ID NO: 2 or 4) sequence. In preferred embodiments of the method, the increasing is at least 3 fold, 5xc3x97, 7xc3x97, 10xc3x97, or more; or the expressing is of a recombinant nucleic acid encoding one or both of the polypeptide and CLF-1 (SEQ ID NO: 12or 13).
The invention further provides a method of screening for a receptor which binds an isolated soluble complex comprising the mature protein portion of SEQ ID NO: 2 or 4, and the mature protein portion of SEQ ID NO: 12 or 13, comprising contacting the complex to a cell expressing the receptor under conditions allowing the complex to bind to the receptor, thereby forming a detectable interaction. Preferably, the interaction results in a physiological response in the cell.
Other embodiments of the invention include, e.g., an isolated soluble complex comprising at least 6 amino acids of the mature protein portion of SEQ ID NO: 2 or 4, and: at least 6 amino acids of the mature protein portion of SEQ ID NO: 12 or 13; or at least 6 amino acids of the mature protein portion of the CNTF-R (SEQ ID NO: 9 or 10). Such complex may, e.g., comprise a recombinant polypeptide of mature SEQ ID NO: 2 or 4; comprise a recombinant polypeptide of mature SEQ ID NO: 12 or 13; comprise a recombinant polypeptide of mature CNTF-R (SEQ ID NO: 9 or 10); comprise both a recombinant polypeptide of mature SEQ ID NO: 2 or 4, and a recombinant polypeptide of mature SEQ ID NO: 12 or 13; comprise both a recombinant polypeptide of mature SEQ ID NO: 2 or 4, and a recombinant polypeptide of mature CNTF-R (SEQ ID NO: 9 or 10); be detectably labeled; be in a buffered solution; or be in a sterile solution. Preferred embodiments include those which: comprise a mature IL-B60 polypeptide (SEQ ID NO: 1 or 2); comprise a mature CLF-1 polypeptide (SEQ ID NO: 12 or 13); comprises a mature CNTF-R ) polypeptide (SEQ ID NO: 9 or 10; exhibit at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 2 or 4; exhibit epitopes from both primate L-B60 (SEQ ID NO: 2) and primate CLF-1 (SEQ ID NO: 12); exhibit epitopes from both primate L-B360 (SEQ ID NO: 2) and primate CNTF-R (SEQ ID NO: 9); not be glycosylated; be attached to a solid substrate; be conjugated to another chemical moiety; or comprise a detection or purification tag, including a FLAG, His6, or Ig sequence.
Kits are provided, e.g., comprising the complex and: a compartment comprising the complex, and/or instructions for use or disposal of reagents in the kit.
Fusion polypeptides are provided, which include, e.g., an isolated or recombinant polypeptide comprising: a first segment comprising at least seven amino acids identical to segments of SEQ ID NO: 2 or 4, and a second segment comprising at least seven amino acids identical to segments of mature SEQ ID NO: 12 or 13; at least two distinct nonoverlapping segments of at least five amino acids identical to segments of mature SEQ ID NO: 2 or 4, and a third segment comprising at least seven amino acids identical to segments of mature SEQ ID NO: 12 or 13; at least one segment comprising at least seven amino acids identical to segments of mature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments of at least five amino acids identical to segments of mature SEQ ID NO: 12 or 13; a first segment comprising at least seven amino acids identical to segments of SEQ ID NO: 2 or 4, and a second segment comprising at least seven amino acids identical to segments of mature primate CNTF-R (SEQ ID NO: 9); at least two distinct nonoverlapping segments of at least five amino acids Identical to segments of mature SEQ ID NO: 2 or 4, and a third segment comprising at least seven amino acids identical to segments of mature primate CNTF-R (SEQ ID NO: 9); or at least one segment comprising at least seven amino acids identical to segments of mature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments of at least five amino acids identical to segments of mature primate CNTF-R (SEQ ID NO: 9). Certain embodiments include those wherein the distinct nonoverlapping segments of identity: include one of at least eight amino acids; include one of at least five amino acids and a second of at least six amino acids: include at least three segments of at least four, five, and six amino acids, or include one of at least twelve amino acids. Other embodiments include those which: comprise a mature IL-B60 sequence; comprise a mature CLF-1 sequence; comprise a mature CNTF-R (SEQ ID NO: 9 or 10) sequence; exhibit at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 2 or 4; have a length at least about 30 amino acids; exhibit epitopes from both primate IL-B60 (SEQ ID NO: 2) and primate CLF-1 (SEQ ID NO: 12); exhibits epitopes from both primate IL-B60 (SEQ ID NO: 2) and primate CNTF-R (SEQ ID NO: 9); are not glycosylated; have a molecular weight of at least 30 kD; be a synthetic polypeptide; be attached to a solid substrate; be conjugated to another chemical moiety; or comprise a detection or purification tag, including a FLAG, His6, or Ig sequence.
Other embodiments include a composition comprising: substantially pure combination of IL-B60 (SEQ ID NO: 2 or4) and CLF-1 (SEQ ID NO: 12 or 13); substantially pure combination of IL-B60 (SEQ ID NO: 2 or 4) and CNTF-R (SEQ ID NO: 9 or 10); a sterile polypeptide described above; or the polypeptide described above and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer, and/or formulated for oral, rectal, nasal, topical, or parenteral administration. A kit is provided comprising a polypeptide as described and: a compartment comprising the polypeptide; and/or instructions for use or disposal of reagents in the kit.
Methods are also provided, e.g., of making an antibody which recognizes a complex as described, comprising inducing an immune response in an animal with the complex; of immunoselecting antibodies, comprising contacting a population of antibodies to a complex as described, and separating antibodies that bind from those which do not bind; or of formulating a composition, comprising admixing a complex as described with a carrier.
Binding compounds are provided, e.g., comprising an antigen binding site from an antibody, which antibody specifically binds a described complex, but not to any of the mature polypeptides of SEQ ID NO: 2, 4, 12, 13, or CNTF-R (SEQ ID NO: 9 or 10). Certain embodiments include those wherein: the binding compound is: in a container; an Fv, Fab, or Fab2 fragment; or conjugated to another chemical moiety; or the antibody: is raised against a substantially pure complex of IL-B60 (SEQ ID NO: 2 or 4) with CLF-1 (SEQ ID NO: 12 or 13); is raised against a substantially pure complex of IL-B60 (SEQ ID NO: 2 or 4) with CNTF-R (SEQ ID NO: 9 or 10); is immunoselected; is a polyclonal antibody; exhibits a Kd to antigen of at least 30 xcexcM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. Additional embodiments include a composition comprising: a sterile binding compound as described, or the binding compound as described and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
With the binding composition are provided a kit comprising the binding compound and: a compartment comprising the binding compound; or instructions for use or disposal of reagents in the kit. Also provided are methods of producing an antigen:antibody complex, comprising contacting under appropriate conditions a primate complex comprising: IL-B60 (SEQ ID NO: 2) and CLF-1 polypeptides (SEQ ID NO: 12); or IL-B60 (SEQ ID NO: 2) and CNTF-R (SEQ ID NO: 9) polypeptides; with an antibody as described, thereby allowing the complex to form. Preferably, in the method, the complex is purified from other cytokines; the complex is purified from other antibody; the contacting is with a sample comprising a cytokine; the contacting allows quantitative detection of the antigen; the contacting is with a sample comprising the antibody; or the contacting allows quantitative detection of the antibody.
Various nucleic acids are provided, e.g., an isolated or recombinant nucleic acid: encoding the amino acid portions described above; encoding the amino acid portions as described, and comprise a segment at least 30 contiguous nucleotides from SEQ ID NO: 1 or 3; which will coexpress a segment of at least seven contiguous amino acids from SEQ ID NO: 2 or 4, and a segment of at least seven contiguous amino acids from SEQ ID NO: 12 or 13; or which will coexpress a segment of at least seven contiguous amino acids from SEQ ID NO: 2 or 4, and a segment of at least seven contiguous amino acids from CNTF-R (SEQ ID NO: 9 or 10). Preferred nucleic acids include those which, e.g.,: encode IL-B60 from a human (SEQ ID NO: 1 or 2); encode CLF-1 from a human (SEQ ID NO: 12); encodes CNTF-R (SEQ ID NO: 9) from a human; are an expression vector; further comprise an origin of replication; comprise a detectable label; comprise synthetic nucleotide sequence; or are less than 6 kb, preferably less than 3 kb. A cell comprising the recombinant nucleic acid is provided, e.g., wherein the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell. Various nucleic acid kits are provided, e.g., comprising the nucleic acid and: a compartment comprising the nucleic acid; a compartment further comprising a primate IL-B60 polypeptide (SEQ ID NO: 2); a compartment further comprising a primate CLF-1 polypeptide (SEQ ID NO: 12); a compartment further comprising a primate CNTF-R (SEQ ID NO: 9) polypeptide; or instructions for use or disposal of reagents in the kit. Methods are also provided, e.g., of making a duplex nucleic acid, comprising contacting such a nucleic acid with a complementary nucleic acid under appropriate conditions, thereby forming said duplex; of expressing a polypeptide, comprising expressing the nucleic acid, thereby producing the polypeptide; or of transfecting a cell, comprising contacting said cell under appropriate conditions with the nucleic acid, thereby transfecting the cell.
In an alternative embodiment, the invention provides an isolated or recombinant nucleic acid which encodes at least 5 contiguous amino acids of SEQ ID NO: 12 or 13, or primate CNTF-R (SEQ ID NO: 9) and: hybridizes under wash conditions of 30 minutes at 30xc2x0 C. and less than 2M salt to the coding portion of SEQ ID NO: 1; or exhibits Identity over a stretch of at least about 30 nucleotides to a primate IL-B60 (SEQ ID NO: 1). Preferred embodiments include: the isolated nucleic acid, wherein: the contiguous amino acids number at least 8; the wash conditions are at 45xc2x0 C. and/or 500 mM salt; or the stretch is at least 55 nucleotides; or the recombinant nucleic acid, wherein: the contiguous amino acids number at least 12; the wash conditions are at 55xc2x0 C. and/or 150 mM salt; or the stretch is at least 75 nucleotides.
The invention particularly provides methods of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a complex comprising mammalian IL-B60 (SEQ ID NO: 2 or 4) and; CLF-1 (SEQ ID NO: 12 or 13); or CNTF-R (SEQ ID NO: 9 or 10). It also provides methods of producing the proteins, e.g., producing a complex described, comprising coexpressing a recombinant IL-B60 (SEQ ID NO: 2 or 4) with a recombinant CLF-1 (SEQ ID NO: 12 or 13) or CNTF-R (SEQ ID NO: 9 or 10); increasing the secretion of an IL-B60 polypeptide (SEQ ID NO: 2 or 4) comprising expressing the polypeptide with CLF-1 (SEQ ID NO: 12 or 13) or CNTF-R (SEQ ID NO: 9 or 10); or increasing the secretion of a CLF-1 (SEQ ID NO: 12 or 13) polypeptide, comprising expressing the CLF-1 (SEQ ID NO: 12 or 13) with an IL-B60 (SEQ ID NO: 2 or 4). Typically, the increasing is at least 3 fold; or the expressing is of a recombinant nucleic acid encoding one or both of the polypeptide and CLF-1 (SEQ ID NO: 12 or 13).
Also provided are methods of screening for a receptor which binds the described complex, comprising contacting the complex to a cell expressing the receptor under conditions allowing the complex to bind to the receptor, thereby forming a detectable interaction. Preferably, the interaction results in a physiological response in the cell.